CAJ | 학술논문

背景：建立一种既可以大量制备，又易于保存并保持较高活性的饲养层细胞是人胚胎干细胞培养研究的重要环节。 　　目的：建立昆明小鼠胚胎成纤维细胞的最佳分离培养方法，评价其用于人胚胎干细胞饲养层研究的可行性。方法：用不同浓度胰蛋白酶分步消化法体外分离和培养昆明小鼠胚胎成纤维细胞，观察其生物学特性，制备胚胎成纤维细胞饲养层，检测人胚胎干细胞在饲养层上培养的生长状态。 　　结果与结论：制备昆明小鼠胚胎成纤维细胞饲养层的最佳胎龄为13.5 d。不同浓度胰蛋白酶分步消化法制备的胚胎成纤维细胞生长状态好，获得的成纤维细胞纯度高，增殖活跃。冻存2周，1，3，6个月内复苏的细胞存活率差异无显著性意义。小鼠胚胎成纤维细胞在第2-4代增殖旺盛，第5代以后细胞增殖活力明显下降。人胚胎干细胞在小鼠胚胎成纤维细胞长期传代后呈典型的未分化形态，碱性磷酸酶和过碘酸-雪夫染色均为阳性。结果表明建立的昆明小鼠胚胎成纤维细胞饲养层分离培养法可为人胚胎干细胞扩增提供稳定、优质的饲养层细胞。
배경：건립일충기가이대량제비，우역우보존병보지교고활성적사양층세포시인배태간세포배양연구적중요배절。 　　목적：건립곤명소서배태성섬유세포적최가분리배양방법，평개기용우인배태간세포사양층연구적가행성。방법：용불동농도이단백매분보소화법체외분리화배양곤명소서배태성섬유세포，관찰기생물학특성，제비배태성섬유세포사양층，검측인배태간세포재사양층상배양적생장상태。 　　결과여결론：제비곤명소서배태성섬유세포사양층적최가태령위13.5 d。불동농도이단백매분보소화법제비적배태성섬유세포생장상태호，획득적성섬유세포순도고，증식활약。동존2주，1，3，6개월내복소적세포존활솔차이무현저성의의。소서배태성섬유세포재제2-4대증식왕성，제5대이후세포증식활력명현하강。인배태간세포재소서배태성섬유세포장기전대후정전형적미분화형태，감성린산매화과전산-설부염색균위양성。결과표명건립적곤명소서배태성섬유세포사양층분리배양법가위인배태간세포확증제공은정、우질적사양층세포。
BACKGROUND:It is important to produce and save a large amount of high-activity feeder cel s for the culture of human embryonic stem cel s. OBJECTIVE:To establish the optimal method for isolation and culture of Kunming mouse embryonic fibroblasts, and to evaluate the feasibility of preparing feeder layers for culture of human embryonic stem cel s. METHODS:Embryonic fibroblasts were isolated and cultured by different concentrations of trypsin from Kunming mouse fetuses in vitro. The biological characteristics and growth rule of mouse embryonic fibroblasts were investigated, and then the feeder layers for human embryonic stem cel s culture were produced. The growth of human embryonic stem cel s on the prepared feeder layer was tested. RESULTS AND CONCLUSION:The optimal fetal age for preparing Kunming mouse embryonic fibroblast feeder layer was 13.5 days. Kunming mouse embryonic fibroblasts at different concentrations grew wel with high purity and active proliferation by trypsin digestion method. There was no significant difference in the survival rate of cel s after cryopreservation for 2 weeks, 1 month, 3 months and 6 months. The cel s were proliferative from the second to fourth passage and declined sharply after the fifth passage. Human embryonic stem cel s which grew on Kunming mouse embryonic fibroblasts feeder layers were stil to remain the typical undifferentiated morphology and were strongly positive for alkaline phosphatase and periodic acid-Schiff after long-term subculture. The mouse embryonic fibroblasts can be used as the stable and high-quality feeder cel s for human embryonic stem cel s.